Linker system for activating surfaces for bioconjugation
The present invention relates to a linker system for activating surfaces for bioconjugation, and particularly to a linker system having a novel hydrophilic spacer group. The inventive linker system may be used for the construction of sensor chips or biochips for the detection of sample biomolecules.
1. A surface carrying a linker system comprising a compound for activating surfaces for bioconjugation having the following general formula (I):
X-[(Y 1 ) i —Q—(Y 2 ) j ] k —Z (I)
wherein:
X is a reactive group capable of covalently binding to a surface and is selected from the group consisting of a SiW 3 group with W being a hydrolyzable atom or group, a group capable of forming free radicals on exposure to light, an anthrathione group or a derivative thereof, an anthraquinone group or a derivative thereof, and a benzophenone group or a derivative thereof;
Z is a reactive group capable of covalently binding to a biomolecule, is capable of nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitutions, and is selected from the group consisting of a diene group, a dienophilic group, an aldehyde group, a hydroxyl group, a carboxylic acid group, an active ester group, an amino group, a thiol group, an aziridine group, an isocyanate group, an isothiocyanate group, an azide group, and a reactive leaving group;
X is not Z;
Y 1 and Y 2 are, independently from each other, CR 1 R 2 ;
R 1 and R 2 are, independently from each other, H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 acyloxy;
i, j, and k are, independently from each other, an integer in the range from 1 to 10;
the total number of C atoms in Y 1 and Y 2 , the C atoms of R 1 and R 2 not included, is in the range of 2 to 100;
Q is a hydrophilic atom or group selected from the group consisting of O, NH, C═O, O—C═O and CR 3 R 4 ;
R 3 and R 4 are, independently from each other, selected from the group consisting of H, OH, C 1 -C 4 alkoxy and C 1 -C 4 acyloxy; and
R 3 and R 4 are not H at the same time;
wherein when Q=NH, Z is not NH 2 ;
wherein when k>1, the Q's for each [(Y 1 ) i —Q—(Y 2 ) j ] k are independently selected from each other; and wherein the linker system is covalently bonded to a biomolecule
wherein said biomolecule is a partner of a specifically interacting system of complementary binding partners;
wherein said specifically interacting system of complementary binding partners is based on nucleic acid/complementary nucleic acid, peptide nucleic acid/nucleic acid, enzyme/substrate, receptor/effector, lectin/sugar, antibody/antigen, avidin/biotin or strept-avidin/biotin interaction; and
wherein said antibody is a polyclonal, monoclonal, chimeric or single-chain antibody or a functional fragment or derivative of such antibodies.
2. Surface according to claim 1 , wherein said surface is selected from the group consisting of a SiO 2 surface of a silicon wafer, glass, quartz, fused silica, gold and a polymer.
3. Medical or diagnostic instrument comprising a surface according to claim 1 .
4. Process for the detection of a biomolecule which is a partner of a specifically interacting system of complementary binding partners, comprising the steps of:
a) contacting a surface with a sample suspected to contain the complementary binding partner,
b) removing non-specifically bound sample components in a washing step, and
c) detecting specifically bound sample components;
wherein the surface carries a linker s stem comprising a compound for activating surfaces for bioconjugation having the following general formula (I):
X—[(Y 1 ) i —Q—(Y 2 ) j ] k —Z (I)
wherein:
X is a reactive group capable of covalently binding to a surface and is selected from the group consisting of a SiW 3 group with W being a hydrolyzable atom or group, a group capable of forming free radicals on exposure to light, an anthrathione group or a derivative thereof, an anthraquinone group or a derivative thereof, and a benzophenone group or a derivative thereof;
Z is a reactive group capable of covalently binding to the biomolecule, is capable of nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitutions, and is selected from the group consisting of a diene group, a dienophilic group, an aldehyde group, a hydroxyl group, a carboxylic acid group, an active ester group, an amino group, a thiol group, an aziridine group, an isocyanate group, an isothiocyanate group, an azide group, and a reactive leaving group;
X is not Z;
Y 1 and Y 2 are, independently from each other, H, C 1 R 2 ;
R 1 and R 2 are, independently from each other, H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 acyloxy;
i, j, and k are, independently from each other, an integer in the range from 1 to 10;
the total number of C atoms in Y 1 and Y 2 the C atoms of R 1 and R 2 not included, is in the range of 2 to 100;
Q is a hydrophilic atom or group selected from the group consisting of O, NH, C═O—C═O and CR 3 R 4 ;
R 3 and R 4 are, independently from each other, selected from the group consisting of H, OH, C 1 -C 4 alkoxy and C 1 -C 4 acyloxy; and
R 3 and R 4 are not H at the same time;
wherein when Q=NH, Z is not NH 2 ;
wherein when k>1, the Q's for each [(Y 1 ) i —Q—(Y 2 ) j ] k are independently selected from each other; and
wherein the linker system is covalently bonded to the biomolecule and said biomolecule is a partner of a specifically interacting system of complementary binding partners.
5. Process according to claim 4 wherein for said detecting, a colored, fluorescent, bioluminescent, chemoluminescent, phosphorescent or radioactive label; an enzyme; an antibody or a functional fragment or derivative thereof, a protein A/gold based system; a biotin/avidin/streptavidin based system; or an enzyme electrode based system is used.
6. The method of claim 4 , wherein said surface comprises a silicon oxide or gold.
7. Process for the isolation of a biomolecule which is a partner of a specifically interacting system of complementary binding partners, comprising the steps of:
a) contacting a surface with a sample suspected to contain the biomolecule complementary binding partner,
b) removing non-specifically bound sample components in a washing step, and, optionally,
c) eluting specifically bound sample components;
wherein the surface carries a linker system comprising a compound for activating surfaces for bioconjugation having the following general formula (I):
X—[(Y 1 ) i —Q—(Y 2 ) j ] k —Z (I)
wherein:
X is a reactive group capable of covalently binding to a surface and is selected from the group consisting of a SiW 3 group with W being a hydrolyzable atom or group, a group capable of forming free radicals on exposure to light, an anthrathione group or a derivative thereof, an anthraquinone group or a derivative thereof, and a benzophenone group or a derivative thereof;
Z is a reactive group capable of covalently binding to the biomolecule, is capable of nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitutions, and is selected from the group consisting of a diene group, a dienophilic group, an aldehyde group, a hydroxyl group, a carboxylic acid group, an active ester group, an amino group, a thiol group, an aziridine group, an isocyanate group, an isothiocyanate group, an azide group, and a reactive leaving group;
X is not Z;
Y 1 and Y 2 are, independently from each other, CR 1 R 2 ;
R 1 and R 2 are, independently from each other, H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 acyloxy;
i, j, and k are, independently from each other, an integer in the range from 1 to 10;
the total number of C atoms in Y 1 and Y 2 , the C atoms of R 1 and R 2 not included, is in the range of 2 to 100;
Q is a hydrophilic atom or group selected from the group consisting of O, NH, C═O, O—C═O and CR 3 R 4 ;
R 3 and R 4 are, independently from each other, selected from the group consisting of H, OH, C 1 -C 4 alkoxy and C 1 -C 4 acyloxy; and
R 3 and R 4 are not H at the same time;
wherein when Q=NH, Z is not NH 2 ;
wherein when k>1, the Q's for each [(Y 1 ) i —Q—(Y 2 ) j ] k are independently selected from each other; and
wherein the linker system is covalently bonded to the biomolecule and the biomolecule is a partner of a specifically interacting system of complementary binding partners.
8. A method of affinity chromatography comprising the steps of:
providing a surface as an affinity matrix; and performing affinity chromatography with the affinity matrix;
wherein the surface carries a linker system comprising a compound for activating surfaces for bioconjugation having the following general formula (I):
X—[(Y 1 ) i —Q—(Y 2 ) j ] k —Z (I)
wherein:
X is a reactive group capable of covalently binding to a surface and is selected from the group consisting of a SiW 3 group with W being a hydrolyzable atom or group, a group capable of forming free radicals on exposure to light, an anthrathione group or a derivative thereof, an anthraquinone group or a derivative thereof, and a benzophenone group or a derivative thereof;
Z is a reactive group capable of covalently binding to a biomolecule is capable of nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitutions, and is selected from the group consisting of a diene group, a dienophilic group, an aldehyde group, a hydroxyl group, a carboxylic acid group, an active ester group, an amino group, a thiol group, an aziridine group, an isocyanate group, an isothiocyanate group, an azide group, and a reactive leaving group;
X is not Z;
Y 1 and Y 2 are, independently from each other, CR 1 R 2 ;
R 1 and R 2 are, independently from each other, H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 acyloxy;
i, j, and k are, independently from each other, an integer in the range from 1 to 10;
the total number of C atoms in Y 1 and Y 2 , the C atoms of R 1 and R 2 not included, is in the range of 2 to 100;
Q is a hydrophilic atom or group selected from the group consisting of O, NH, C═O, O—C═O and CR 3 R 4 ;
R 3 and R 4 are, independently from each other, selected from the group consisting of H, OH, C 1 -C 4 alkoxy and C 1 -C 4 acyloxy; and
R 3 and R 4 are not H at the same time;
wherein when Q=NH, Z is not NH 2 ;
wherein when k>1, the Q's for each [(Y 1 ) i —Q—(Y 2 ) j ] k are independently selected from each other; and
wherein the linker system is covalently bonded to a biomolecule and the biomolecule is a partner of a specifically interacting system of complementary binding partners.
9. A method of detecting a biomolecule comprising the steps of:
providing a sensor chip or biochip comprising a surface; and detecting a biomolecule with the sensor chip or biochip;
wherein the surface carries a linker system comprising a compound for activating surfaces for bioconjugation having the following general formula (I):
X—[(Y 1 ) i —Q—(Y 2 ) i ] k —Z (I)
wherein:
X is a reactive group capable of covalently biding to a surface and is selected from the group consisting of a SiW 3 group with W being a hydrolyzable atom or group, a group capable of forming free radicals on exposure to light, an anthrathione group or a derivative thereof, an anthraquinone group or a derivative thereof, and a benzophenone group or a derivative thereof;
Z is a reactive group capable of covalently binding to a biomolecule, is capable of nucleophilic substitution reactions, nucleophilic addition reactions, Diels-Alder reactions or radical substitutions, and is selected from the group consisting of a diene group, a dienophilic group, an aldehyde group, a hydroxyl group, a carboxylic acid group, an active ester group, an amino group, a thiol group, an aziridine group, an isocyanate group, an isothiocyanate group, an azide group, and a reactive leaving group;
X is not Z;
Y 1 and Y 2 are, independently from each other, CR 1 R 2 ;
R 1 and R 2 are, independently from each other, H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy or C 1 -C 4 acyloxy;
i, j, and k are, independently from each other, an integer in the range from 1 to 10;
the total number of C atoms in Y 1 and Y 2 , the C atoms of R 1 and R 2 not included, is in the range of 2 to 100;
Q is a hydrophilic atom or group selected from the group consisting of O, NH, C═O, O—C═O and CR 3 R 4 ;
R 3 and R 4 are, independently from each other, selected from the group consisting of H, OH, C 1 -C 4 alkoxy and C 1 -C 4 acyloxy; and
R 3 and R 4 are not H at the same time;
wherein when Q=NH, Z is not NH 2 ;
wherein when k>1, the Q's for each [(Y 1 ) i —Q—(Y 2 ) j ] k are independently selected from each other; and
wherein the linker system is covalently bonded to a biomolecule and the biomolecule is a partner of a specifically interacting system of complementary binding partners.